Polycosanols from Ericerus pela wax

ABSTRACT

The present invention provides a novel method for the preparation of a unique profile of primary aliphatic alcohols, having 24 to 30 carbon atoms, from the wax secreted by the insect  Ericerus pela . Included in the present invention is the composition of matter, referred to herein as “polycosanol” produced by the method of this invention. The polycosanol composition is comprised primarily of the four primary aliphatic alcohols, tetracosanol, hexacosanol, octacosanol and triacontanol. Further included in this invention is the use of said composition of matter for the prevention and treatment of obesity, syndrome X, diabetes, hypercholesterolemia, atherosclerotic complications, ischemia and thrombosis.

RELATED APPLICATIONS

This application is a continuation in part of U.S. application Ser. No.10/658,881, filed Sep. 9, 2003, now U.S. Pat. No. 6,822,004, which is adivisional of U.S. application Ser. No. 10/356,676, filed Jan. 31, 2003,now U.S. Pat. No. 6,683,116 each of which is entitled “Polycosanols fromEricertis Pela Wax,” and each of which is incorporated herein byreference in its entirety.

FIELD OF THE INVENTION

The present invention relates generally to a method for the generationof a composition of matter comprised of a unique profile of primaryaliphatic alcohols, having 24 to 30 carbon atoms, from a novel naturalsource—a wax secreted by the Chinese wax soft scale insect, Ericeruspela, which belongs to the family Coccidae. The present inventionincludes the composition of matter, referred to herein as“polycosanol(s)” produced by the method of the invention. Thepolycosanol composition produced according to the method of thisinvention is comprised of primarily four primary aliphatic alcohols,tetracosanol, hexacosanol, octacosanol and triacontanol. The inventionalso includes the use of this composition of matter for the preventionand treatment of obesity, syndrome X, diabetes, hypercholesterolemia,atherosclerotic complications, ischemia and thrombosis.

BACKGROUND OF THE INVENTION

Polycosanols are a class of primary aliphatic alcohols having 20 to 40(C20-C40) carbon atoms. They are widely distributed in germs, kernelsand other components of nuts, seeds, fruits and cereals (Kawanishi etal. (1991) J. Amer. Oil Chemist Soc. 68:869-872), in Greek olive oils(Dimitrios et al. (1983) Grasas Aceites 34:402-404) and in apple wax(Belding et al. (1993) Hertscience 28:90). Polycosanols are alsopresent, in very small amounts (less than 0.1%), in wheat grain, in theform of the long chain alkyl esters of fatty acids. The major compoundspresent in wheat grain include, palmitoyl hexacosanol and arachidoyl,palmitoyl and behenoyl tetracosanol (Ohnishi et al. (1986) Cereal Chem63:193-196).

Polycosanols, both as the free alcohols and the esters of fatty acids,have been isolated from many different genera and species of plants,including from the species Achillea biebersteinii (Oskay and Yeslada(1984) J. Nat. Prod. 47:742), Calamagrostis arundinacea (Solberg (1976)Acta Chem. Scand Ser. B. Org. Chem. Bioche. 30:786-787), Emiliasonchifolia (Srinivasan and Subramanian, (1980) Fitoterapia 51:241-244),Heliotropium digynum (Ismail et al. (1984) Fitoterapia 55:110-112),Hypericum perforatum (Brondze (1983) J. Nat. Prod. 46:940-941),Tragopogon orientalis (Krzaczek et al. (1988) Acta Soc. Bot. Pol.57:85-92), and from the genera of Triceae (Tulloch (1981) Can. J. Bot.58:2602-2615). Polycosanols have also been isolated from various partsof many different genera and species of plants, including from the barkof various Acacia species (Banerji and Nigram (1980) J. Indian Chem.Soc. 57:1043-1044); from the stem of Anisomeles indica (Dobhal et al.(1988) Fitoterapia 59:155); from the leaves of Cordia rothii (Behari etal. (1980) Acta Cienc. Indica Chem. 6:226-228), Hibiscus cannabinus(Makhsudova (1979) Chem. Nat. Compd. 15:186) and Holigarna arnottiana(Prakash and Banerji (1979) Fitoterapia 50:265-266); from the roots ofTalinum paniculatum (Komatsu et al. (1982) Yakugaku Zasshi 102:499-502);from the leaves and roots of Gymnosporia Montana (Kumar andSrimannarayana (1981) J. Nat. Prod. 44:625-628); from the aerial partsof Cymbopogon citrates (Olaniyi et al. (1975) Planta Medica 28:186-189),E. merifolia (Baslas and Agarwal (1980) Indian J. Pharm. Sci. 42:66-67)and E. Peplus (Rizk et al. (1980) Fitoterapia 51:223-228), Portulacasuffruticosa L. (Joshi et al. (1987) Herba Pol. 33:71-74) and Youngiadenticulate (Arai et al. (1982) J. Pharm. Soc. Jp. 102:1089-1091); andfrom the heartwood of Melia birmanica (Banerji and Nigam, (1981)Fitoterapia 52:3-4). Polycosanols have also been isolated from carnaubawax from exudates of the leaves of the palm tree Copernicia cerifera(Pinto and Bento (1986) Rev Soc Bras Med Trop 19:243-5), from the leafwax of Euphorbia helioscopia (Nazir et al. (1993) Xeischrift furNaturforschung. Section C Biosciences 48:5-9), from epicuticular waxesof genera of Gramineae (Tulloch (1981) Can. J. Bot. 59:1213-1221), andfrom and the latex of Euphorbia pseusocactus (Awad et al. (1993)Fitoterapia 64:553) and Euphorbia thymifolia (Agarwal and Maslas (1981)Indian J. Pharma. Sci. 43:182-183). Finally, polycosanols have beenisolated from the Korean indigenous plant Echinosophora koreensis (Kangand Kim (1987) Arch Pharmacal Res. 10:67-68).

Bertholet has described a method for preparing polycosanol compositionsby means of the saponification of plant wax from rice bran wax, carnaubawax and jojoba oil. (Bertholet, U.S. Pat. No. 5,159,124 (1991)). In themethod described by Bertholet, the plant wax was first dissolved in anorganic water immiscible solvent, such as butanol or pentanol, and thenhydrolyzed using an aqueous solution of an alkaline earth metalhydroxide. The fatty acid by products of the saponification reaction aresoluble in the alkaline aqueous layer and the polycosanol alcoholproduct remains in the organic layer, which contains <10% fatty acidsand >90% alcohols. The overall yield of the reaction was approximately50%. The composition of the polycosanol product is dependent on theorigin of the plant wax.

N-hexacosanol has been isolated from wool wax hydrosylate mixtures usinggel permeation chromatography (Steel et al. (1999) InternationalPublication No. WO 99/48853).

Polycosanol compositions isolated from rice bran wax have beenformulated with phytosterol from vegetable oil and used for reducingcholesterol levels. The aliphatic alcohol profile of this material isapproximately 23-33% total polycosanol. Triacontanol is the majorcompound (8-9%), followed by octacosanol (5-6%) and tetracontanol,hexacosanol, dotriacontanol and tetratriacosanol (2-5% each). (SorkinJr. (1999) U.S. Pat. No. 6,197,832, and Sorkin Jr. (1998) U.S. Pat. No.5,952,393). Octacosanol isolated from Sinach has been formulated withother ingredients as a nutritional powder for boosting energy. (GaynorU.S. Pat. No. 5,744,187 (1996)).

Sugar cane provides a major natural source of commercial polycosanolproducts (Ali et al. (1979) Egypt J. Pharm. Sci. 18:93-99). The longchain aliphatic alcohols are located primarily in the wax layer of sugarcane, with octacosanol being the predominant compound (Nagata et al.(1994) Breeding Sci. 44:427-429) (See Table 1, below). Aliphaticalcohols from sugar cane wax can be extracted directly with asupercritical fluid, an organic solvent or an alcohol to obtain amixture with octacosanol (7-10%) and triacontanol (0.4-1%) as the majorcomponents (Inada et al. (1986) U.S. Pat. No. 4,714,791). A mixture ofhigher primary aliphatic alcohols, having from 24 to 34 carbon atoms hasbeen obtained by saponification of sugar cane wax. (Laguna et al. (1996)U.S. Pat. No. 5,856,316). The saponification reaction described includedmelting the sugar cane wax, forming a homogeneous phase with an alkalineearth hydroxide (5-30%), extracting with an organic solvent andrecrystallizing from an organic solvent. The profile of the materialincluded octacosanol as the major component (60-70% content), followedby triacontanol (10-15%), hexacosanol (5.5-8.5%), dotriacontanol (4-6%),heptacosanol (2-3.5%), tetratriacontanol (0.4-2.0%), nonacontanol(0.4-1.2%) and tetracosanol (0.5-1.0%). This material has beenformulated with acetylsalicylic acid and used for the treatment ofhypercholesterolemia, atherosclerotic complications, gastric ulcers andto improve male sexual activity. (Laguna et al. (1996) U.S. Pat. No.5,856,316).

Ericerus pela, which belongs to the family Coccidae (Ben-Dov andHodgson, (1997) Soft scale insects; their biology, natural enemies andcontrol. Vol. 7A. Elsevier Science Publishers, Amsterdam), is an insectindigenous to southern China, having the common name white wax scale.(Zhang (1987) Scientia Silvae Sinica 23:383-385, Cen and Ji (1988)Insect Knowledge 25:230-232). This insect has a high economic value inChina (Chen (1999) World Forest Res. 12:46-52), due to its ability toproduce wax and its high nutritional value (Zhao et al. (2001)Entomological Knowledge 38:216-218). The female lays over 7000 eggs onaverage (Park et al. (1998) Korea J. Applied Entomology 37:137-142) andegg hatching is directly related to wax production (Chen et al. (1997)Forest Res. 10:149-153). The reproductive capability of Ericerus pelacan be impacted by sex ratio, lifespan, habitat and other ecologicalconditions. (Zhang et al. (1993) Entomological Knowledge 30:297-299).The eggs from this insect contain a high percentage of proteins (40-55%)and amino acids (30-50%) and are nutritious and safe for humanconsumption. (Ye et al. (2001) Forest Res. 14:322-327). The insect canbe raised on 200 different species of host plants belonging to 98 generaand 36 families. (Chen and Li (2001) Forest Res. Beijing 14:100-105).The host plants provide not only their habitat and reproductive sites,but also serve as their food source. (Chen et al. (1997) Forest Res.10:415-419). The average amount of wax production is affected by thehost plant species (Chen et al. Forest Res. 11:285-288), geographicvarieties of the insect (Chen et al. (1998) Forest Res. 11:34-38) andclimate conditions, particularly temperature, dryness and intensesunshine. (Liu et al. (1998) Forest Res. 11:508-512). Ericerus pela hasbeen produced in commercial forest plantations and the conditions andvalue of crop production have been reported. (Liu et al. (1996) ForestRes. 9:296-299).

The wax from Ericerus pela is secreted from the wax gland of both maleand female insects. (Tan and Zhong (1992) Zoological Res. 13:217-222).The composition of the insect wax has been analyzed by GC/MS anddetermined to be hexacosyl hexacosanoate (55.16%), hexacosyltetracosanoate (22.36%) and hexacosyl octacosanol (16.65%). (Takahashiand Nomura (1982) Entomol. Gen. 7:313-316). The wax has traditionallybeen used for bleeding, pain relief, wound healing, coughing anddiarrhea. (Li (1985) World Animal Review 55:26-33). Saturated long chainfatty alcohols have also been found in other insects, including Drosichacorpulenta (Hashimoto and Kitaoka (1983) Appl. Entomol Zool.17:453-459).

Bee wax also contains a significant quantity of long chain primaryalcohols in both the free and esterified forms. Polycosanol compositionsisolated from bee wax contain 24 to 34 carbon atoms (C24-C34) comprisedof tetracosanol (9-15%), hexacosanol (12-18%), octacosanol (13-20%),triacontanol (20-30%) and dotriacontanol (13-21%). (Hernandez et al.,U.S. Pat. No. 6,235,795 (1994)). Bee wax, formulated with olive oil,β-sitosterol and an extract from Coptis chinensis, has been used for thetreatment of diaper rash (Niazi, U.S. Pat. No. 6,419,963 (2001)) and asa pharmaceutical and cosmetic carrier (Xu, U.S. Pat. No. 5,817,322(1996)). Polycosanols isolated from bee wax also show anti-ulcer andanti-inflammatory activity. (Mas (2001) Drugs of the Future, 26:731-744;Carbajal et al. (1996) J. Pharmacy and Pharmacol. 48:858-860; Hernandezet al., U.S. Pat. No. 6,235,795 (1994)).

Polycosanol compositions isolated from bee wax upon saponificationcontain primarily octacosanol (13.0-20.0%), triacontanol (20-30%),dotriacontanol (13-21%), hexacosanol (12-18%), tetracosanol (9-15%) andtetratriacontanol (1.5-3.5%). (Hernandez et al., U.S. Pat. No. 6,465,526(2000)). In the method reported by Hernandez et al., the saponificationreaction was performed in the homogeneous phase using a 4-7:1 wax:baseratio. After hydrolysis, the polycosanols were extracted with organicsolvents to produce a product that contained 80-98% total polycosanolsin a yield of approximately 30% from bee wax. (Hernandez et al., (1994)U.S. Pat. No. 6,235,795). As noted above, this material showed bothanti-ulcer and anti-inflammatory activity. Polycosanol compositionsobtained from the saponification of bee wax have also been formulatedwith acetyl salicylic acid for use in the treatment ofhypercholesterolemia, atherosclerotic complications, gastric ulcers andto improve male sexual activity (Granja et al., U.S. Pat. No. 5,663,156(1994)).

The polycosanols in bee wax have also been extracted directly withorganic solvent without saponification. (Perez, U.S. Pat. No. 6,225,354(1999)). This material contained octacosanol (30-60%), triacosanol(16-26%), dotriacontanol (13-22%) and hexacosanol (7-12%) as the majorcomponents and has been shown to be effective in the treatment andprevention of hypercholesterolemia related diseases. (Perez, U.S. Pat.No. 6,225,354 (1999)).

Polycosanol compositions isolated from sugar cane have been shown tolower cholesterol levels in both animal and human models. (Menedez etal. (2000) Br. J. Clin. Pharmacol. 50:255-262; Arruzazabala et al. Braz.J. Med. Biol. Res. 33:835-840; Crespo et al. (1999) Int. J. Clin.Pharmacol. 19:117-127; Gouni-Berthold and Berthold (2002) Am. Heart J.143:356-365; Alcocer et al. (1999) Int. J. Tissue React 21:85-92).Modulation of 3-hydroxy-3-methylglutaryl-Coenzyme A (HMG-CoA) reductasewas observed in a celline model, but not in a pure enzyme inhibitionassay. (Menendez et al. (2001) Arch. Med. Res. 32:8-12). Instead ofinhibiting of 3-hydroxy-3-methylglutaryl-Coenzyme A (HMG-CoA) reductase,as most cholesterol lowering drugs, polycosanol may have differentmechanism of action, such as the down regulation of HMG-CoA reductaseproduction in gene expression and/or at the proteomic level. (McCarty(2002) Med. Hypotheses 59:268).

Older patients with hypertension and Type II hypercholesterolemia,treated with polycosanol compositions isolated from sugar cane at adosage of 20 mg/day for twelve months, showed significantly decreasedTC, LDL, LDL/HDL and TC/HDL levels, and increases HDL levels. (Castanoet al. (2002) Drug R D. 3:159-172; Castano et al. (2001) Int. J. Clin.Pharmacol. 21:43-57). Even at a dosage of 5-10 mg, polycosanolcompositions isolated from sugar cane showed a significant benefit inhypercholesterolemia postmenopausal women (Mirkin et al. (2001) Int. J.Clin. Pharmacol. 21:31-41; Castano et al. (2000) Gynecol. Endocrinol.14:187-195) and in high coronary risk older patients (Castano et al.(2001) J. Geontol A Biol. Sci. Med. Sci. 56:M186-192; Castano et al.(1999) Int. J. Clin. Pharmacol. 19:105-116). In summary, it has beenproposed that polycosanol compositions isolated from sugar cane couldpotentially provide a new treatment for cardiovascular disease withequal or better clinical output than simvastatin, pravastatin,lovastatin, probucol and acipimox. (Janikula (2002) Altern. Med. Rev.7:203-217).

Polycosanol compositions have also been shown to exhibit anti-thrombiceffects (Carbajal et al. (1998) Pharmacol. Res. 38:89-91), withsignificant inhibition of platelet aggregation (Arruzazabala et al.(1993) Thromb Res. 69:321). Additionally, unlike aspirin polycosanol didnot affect the platelet anti-aggregating enzyme PGI2, but ratherinhibited platelet aggregating enzyme thromboxane B2 (TXB2). (Carbajalet al. (1998) Prostaglangins Leukot. Essent Fatty Acids 58:61-64). Thismakes a combination therapy of polycosanol with aspirin an attractiveoption. (Arruzazabala et al. (1997) Pharmacol. Res. 36293-297). Forother reports on the anti-thrombic effects of polycosanol compositionssee Arruzazabala et al. (2002) Clin. Exp. Pharmacol. 29:891-7; Janikula(2002) Alternative Medicine Review 7:203-217; and Stusser et al. (1998)Int J Clin Pharmacol Ther 36(9):469-73). For reports on othercardiovascular benefits of polycosanol compositions see Noa et al.(1997) J. Pharm. Pharmacol. 49:999-1002; Noa et al. (2001) Pharmacolo.Res. 43:31-37; Molina et al. Braz. J. Med. Biol. Res. 32:1269-1276;Janikula (2002) Alternative Medicine Review 7:203-217; and Menendez etal. (2002) Can J Physiol Pharmacol. 80:13-21.

Polycosanol(s) and polycosanolic acids have also been reported to beeffective as nutritional and therapeutic preparations for the preventionand treatment of aging and related conditions, such as, atherosclerosis,hypertension, diabetes, tumors, obesity, overweight,hypertriglyceridemia, hypercholesterolemia, as well as other conditions.(Pistolesi, WO 02/052955 (2001)). There are a numerous other reporteduses of individual polycosanols and mixtures thereof in the literature.This provides a significant incentive to develop new sources containingnovel polycosanol compositions of matter, which would be expected tohave different pharmacological effects and strengths. The multitude ofuses for the individual alcohols and mixtures thereof, also provides asignificant incentive to develop improved methods for isolating thesecompounds.

Polycosanol has been determined to be safe at a dosage of up to 500mg/kg/day, which is 1500 times greater than the standard human dosage of20 mg/day. Rats treated with a dosage of 500 mg/kg/day for 12 to 24months exhibited no signs of toxicity or carcinogenesis resulting fromtreatment with polycosanols. (Aleman et al. (1995) Food Chem. Toxicol.33:573-578). Dogs given 180 mg/kg/day for one year showed no sideeffects resulting from the composition (Mesa et al. (1994) Toxicol.Lett. 73:81-90) and monkeys given 25 mg/kg/day for 54 months showed nosigns of adverse effects (Rodrigurz et al. (1994) Food Che. Toxicol.32:565-575). In reproductive and fertility studies, polycosanolcompositions exhibited no adverse effects on fertility, reproduction anddevelopment in rats fed up to 500 mg/kg/day for two weeks before mating,throughout pregnancy, and 21 days into lactation, and in male rats given500 mg/kg/day for 60 days prior mating (Rodriguez and Garcia (1998)Teratog. Carcinog. Mutagen. 18:1-7). Rabbits treated with a dosage of1000 mg/kg/day during pregnancy showed no evidence of teratogenic andembryonic toxicity. The tissue distribution of polycosanol in animalmodels has been reported by Kabir and Kimura. ((1995) Ann. Nutr. Metab.39:279-284 and (1993) 37:33-38). Polycosanol has been shown to be stablein 10 mg tablets for up to nine months (Cabrera et al. (2002) Boll.Chim. Farm. 141:223-229) with no interaction with excipients (Cabrera etal. (2002) Boll. Chim. Farm. 141:138-142).

Cholestin™, a dietary supplement from Pharmanex, contains octacosanolisolated from the wax of honey bees. This product has been shown topromote healthy cholesterol levels by inhibiting the production ofcholesterol in the liver. LesstanoL™ brand from Garuda International,Inc. contains natural octacosanol (95%) isolated from sugar cane orvegetable waxes. TwinLab Octacosanol Plus is derived from spinach, asuperior and all natural source of octacosanol. Octacosanol in Nature'sWay's products is a naturally occurring substance found in sugar cane,wheat germ oil, spinach, and other natural sources. Octacosanol fromViable Herbal Solutions is the active ingredient in wheat germ oil andis used to increase endurance, stamina and vigor. Applicant is not awareof any reports regarding the production polycosanol compositions fromEricerus pela wax.

Sierra et al. has developed a gas chromatographic (GC) method fordetermining the fatty alcohol content of film-coated tablets. (Sierra etal. (2002) J. AOAC Int. 85:563-566). 1-Octacosanol in rat plasma hasbeen quantified after solid phase extraction and derivatization using acapillary GC method developed by Marrero and Gonzalez ((2001) J.Chromatogr. B Biomed. Sci. Appl. 762:43-49). The polycosanols werederivatized with N-methyl-N-trimethylsilyltrifluoroacetamide. Thegeneral physical characteristics of sugar cane polycosanols have beenreported by Uribarri et al. ((2002) Drug Dev. Ind. Pharm. 28:89-93).

It is an objective of this invention to provide a mixture of higherprimary aliphatic alcohols, referred to herein as “polycosanol(s)”having a unique chemical composition profile.

It is another objective of this invention to provide an improved methodfor obtaining a highly pure mixture of higher primary aliphaticalcohols.

SUMMARY OF THE INVENTION

The present invention includes a novel method for the preparation of aunique profile of primary aliphatic alcohols, having 24 to 30 carbonatoms, from the wax secreted by the insect Ericerus pela. Included inthe present invention is the composition of matter, referred to hereinas “polycosanol” produced by the method of this invention. Thepolycosanol composition produced by the method of this invention iscomprised primarily of two major components, the primary aliphaticalcohols, hexacosanol and octacosanol, with two minor components, theprimary aliphatic alcohols, tetracosanol and triacontanol. Furtherincluded in this invention is the use of said composition of matter forthe prevention and treatment of obesity, syndrome X, diabetes,hypercholesterolemia, atherosclerotic complications, ischemia andthrombosis.

The method of the present invention is comprised of the steps of (a)hydrolyzing the melted wax obtained from the insect with a base; (b)neutralizing the basic hydrosylate obtained from step (a) to yield alower purity composition of matter comprised of polycosanols; and (c)optionally extracting the hydrosylate without neutralization with anorganic solvent to obtain a higher purity polycosanol composition. Inanother embodiment of the instant invention, the method furthercomprises the step of (d) purifying the hydrolyzed product obtained instep (c) by recrystallization. The method of this invention can beextended to the isolation and purification of polycosanol compositionsfrom any source of wax, in particular from any source of insect wax.

The present invention includes the novel mixtures of primary long chainaliphatic alcohols (referred to herein as “polycosanol”) prepared andisolated by the methods of this invention. The long chain aliphaticalcohol portions of the polycosanol compositions prepared and isolatedby the methods of this invention comprise 1-hexacosanol (20%-50%),1-octacosanol (15%-45%), 1-triacontanol (2%-10%) and 1-tetracosanol(approximately 1%-9%). The total amount of long chain aliphatic alcoholsin these compositions can be anywhere in the range of 35% to 100%,depending on the level of purification of the crude extract obtainedfrom step (b) of the described method. In one embodiment, thecomposition of matter is isolated following hydrolysis of the wax andneutralization with no further purification. This composition iscomprised of approximately 35-55% of the long chain primary aliphaticalcohols of interest. The major components of this composition are:1-hexacosanol (˜20-30%) and 1-octacosanol (˜15-25%) and the minorcomponents are: 1-triacontanol (˜2-4%) and 1-tetracosanol (˜1-3%). Inanother embodiment, the composition of matter is isolated followinghydrolysis of the wax and further purification via extraction of thepolycosanols under basic conditions with an organic solvent. In thisembodiment the composition of matter is comprised of approximately75-100% of the long chain primary aliphatic alcohols. The majorcomponents of this composition are 1-hexacosanol (˜30-50%),1-octacosanol (˜25-45%), 1-triacontanol (˜4-10%) and 1-tetracosanol(˜3-9%). In yet another embodiment, the composition can be even furtherpurified by recrystallization. As noted above, the alcoholic mixturesobtained from Ericerus pela wax in accordance with the present inventioncan be distinguished from all other currently known sources ofpolycosanols. Table 1, below highlights the differences between thecompositions isolated from sugar cane and bee wax, which are currentlythe major commercially available sources of polycosanols, with thecomposition isolated according to the method of this invention.

The present invention also includes the use of the polycosanolcompositions isolated by the method of this invention for the preventionand treatment of obesity, syndrome X, diabetes, hypercholesterolemia,atherosclerotic complications, ischemia and thrombosis. These novelcompositions are expected to have different pharmacological effects andstrengths. The compositions can be formulated in a pharmaceuticalcomposition, foodstuff or dietary supplement and administered to humansand other animals.

The present invention provides not only a novel source of polycosanolsand the resulting novel compositions of matter, but also provides animproved method for extracting these compounds from a wax. Preferredlevels of certain operational parameters in the extraction andpurification process have been discovered which lead to furtherenhancement of the purity level of the isolated alcohols and enhancementof the percent recovery of the alcohols from Ericerus pela wax. Theseoperational parameters include optimized solvent volume and quantity ofbasic solution in the hydrolysis process; and extraction of the basicpowder of crude polycosanol with lower polarity organic solvents thanthose used in current methods, which is critical to obtaining higherquality insect polycosanol.

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory onlyand are not restrictive of the invention as claimed.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts the chemical structure of a representative long chain(C22) aliphatic alcohol and a representative structure of an esterifiedfatty acid.

FIG. 2 illustrates the gas chromatographic (GC) profile of eightpolycosanol standards.

FIG. 3 illustrates the gas chromatographic profile of wax from theinsect Ericerus pela before hydrolysis with no polycosanol present.

FIG. 4 depicts the gas chromatographic profile of wax from the insectEricerus pela after hydrolysis. The hydrosylate contains tetracosanol(7.7 min.), hexacosanol (8.7 min.), octacosanol (10 min.) andtriacontanol (11.6 min.).

FIG. 5 depicts the linear range of tetracosanol in the GC analysis.

FIG. 6 illustrates the linear range of hexacosanol in the GC analysis.

FIG. 7 depicts the linear range of triacontanol in the GC analysis.

FIG. 8 depicts the linear range of octacosanol in the GC analysis.

DETAILED DESCRIPTION OF THE INVENTION

The present invention includes a method for the preparation of a uniqueprofile of primary aliphatic alcohols, having 24 to 30 carbon atoms,from the wax secreted by the insect Ericerus pela. Included in thepresent invention is the composition of matter, referred to herein as“polycosanol” produced by the method of this invention. The polycosanolcomposition produced by the method of this invention is comprisedprimarily of only four primary aliphatic alcohols, tetracosanol,hexacosanol, octacosanol and triacontanol. Further included in thisinvention is the use of said composition of matter for the preventionand treatment of obesity, syndrome X, diabetes, hypercholesterolemia,atherosclerotic complications, ischemia and thrombosis.

Various terms are used herein to refer to aspects of the presentinvention. To aid in the clarification of the description of thecomponents of this invention, the following definitions are provided.

It is to be noted that the term “a” or “an” entity refers to one or moreof that entity. As such, the terms “a” or “an,” “one or more” and “atleast one” are used interchangeably herein.

As used herein the term “higher primary aliphatic alcohols” refers toprimary aliphatic alcohols having 24 to 30 carbon atoms (C24-C30). Morespecifically, the term higher primary aliphatic alcohols refers to thefour alcohols—tetracosanol, hexacosanol, octacosanol and triacontanol.

As used herein the term “polycosanol” refers to the mixture of higherprimary aliphatic alcohols derived from the hydrolysis of the wax of theinsect Ericerus pela.

“Therapeutic” as used herein, includes treatment and/or prophylaxis.When used, therapeutic refers to humans, as well as, other animals.

“Pharmaceutically or therapeutically effective dose or amount” refers toa dosage level sufficient to induce a desired biological result. Thatresult may be the delivery of a pharmaceutical agent, alleviation of thesigns, symptoms or causes of a disease or any other desired alterationof a biological system.

A “host” is a living subject, human or animal, into which thecompositions described herein are administered.

Note, that throughout this application various citations are provided.Each citation is specifically incorporated herein in its entirety byreference.

The method of the present invention for preparing a unique profile ofprimary aliphatic alcohols is comprised of the steps of (a) hydrolyzingthe melted wax obtained from the insect Ericerus pela with a basesolution; (b) neutralizing the basic hydrosylate obtained from step (a)to yield a composition of matter comprised of polycosanols; and (c)optionally extracting the hydrosylate without neutralization with anorganic solvent to obtain a higher purity polycosanol composition. Inanother embodiment of the instant invention, the method of the inventionfurther comprises the step of (d) purifying the hydrolyzed productobtained in step (c) by recrystallization.

The method of the present invention is based on the homogeneous phasehydrolysis/saponification of the wax isolated from the insect Ericeruspela. The wax is first melted at a temperature of between 80° C. to 100°C. and then treated with a base in either an aqueous or an alcoholicsolution. The ratio of the amount of wax to the volume of solvent isvery critical to ensure the completion of the hydrolysis reaction. Instead of less than a 1:1 ratio of wax:solvent, the current inventionuses at least a 1:1 and up to a 1:12 ratio of wax:solvent. In theabsence of sufficient solvent, the free polycosanols will begin tosolidify during the middle of the reaction and will trap a significantamount of the non-hydrolyzed esters in the solid. This will result in alow yield of highly impure final product, due to the high solubility ofthese non-hydrolyzed esters in organic solvents. Any base used by thoseof skill in the art can be used to perform the hydrolysis. In apreferred embodiment, the base is selected from an alkaline earthhydroxide including, but not limited to NaOH, KOH or CaOH.

As noted above, the hydrolysis can be performed in an aqueous solutionor an alcoholic solution. In a preferred embodiment, the hydrolysis isperformed in an alcoholic solution because the wax is more soluble inalcohol than in water. Any primary, secondary or tertiary alcohol havingfrom one to ten carbon atoms can be used as the solvent including, butnot limited to methanol, ethanol, propanol and n-butanol.

The concentration of the hydroxide solution must be selected such thatthe ratio in weight of the corresponding hydroxide to that of the wax tobe processed is greater than 5%. In a preferred embodiment, the weightratio of hydroxide to wax is from about 8% to 40%, most preferably theweight ratio of hydroxide to wax is about 25%. The use of a greaterpercentage of hydroxide to perform the hydrolysis reaction is novel tothe current application. It will not only maintain higher pH value thatleads to the completion of the hydrolysis reaction, but also plays avery critical role in obtaining high purity polycosanol products. Thesaponification reaction is allowed to proceed for a time period of atleast 30 minutes to 40 hours. Preferably, the reaction is allowed toproceed for a time period of about 2 to 6 hours. Thehydrolysis/saponification process is facilitated by mechanic agitationand heating both with and without pressurization. The reactiontemperature range is between 50° C. to 200° C. In a preferredembodiment, the reaction is performed at 100° C. and ambient pressurefor approximately six hours.

Upon completion of the hydrolysis the hydrosylate is neutralized and theresidual solvent is removed to provide a solid residue comprised of acomposition of matter of low purity polycosanols. The hydrosylate can beneutralized using any organic or inorganic acid known to one of skill inthe art to perform such a neutralization. In one embodiment, the acid isselected from the group including, but not limited to acetic acid,sulfuric acid, phosphoric acid, choleric acid, nitric acid andhydrochloric acid. In one embodiment of the invention, the hydrosylatereaction mixture is adjusted to a pH of about 1 to 6. Afterneutralization, the residual solvent is removed using a methodincluding, but not limited to filtration, centrifugation, decanting,evaporation, concentration, crystallization or a combination thereof.

The solid residue comprised of a composition of matter of low puritypolycosanols obtained without neutralization can optionally be furtherpurified by extraction with an organic solvent. Contrary to reportedmethods, which extract the polycosanol compositions subsequent toneutralization of the reaction mixture, the current inventionintentionally maintains the strongly basic conditions by significantlyincreasing the amount of base used in the hydrolysis reaction andextracting without neutralization. The excess base remaining aftercompletion of the hydrolysis, keeps the higher primary aliphatic fattyacids, mainly hexacosanic acid in the form of their sodium salts. Sincethe water solubility of hexacosanic acid sodium salt is much higher thanits solubility in organic solvents, this will prevent hexacosanic acidfrom extracting into the organic solvent. This results in a polycosanolproduct that has a much greater purity.

The extraction can be performed using any method of extraction known tothose of skill in the art. In one embodiment the extraction is performedby liquid-liquid partition or solid-liquid extraction. The extractionsolvent is selected from the group of organic solvents including, butnot limited to hydrocarbons having 6 to 9 carbon atoms, such as,pentane, hexane, heptane, octane or a mixture of hydrocarbons such aspetroleum ether; ketones having 3 to 8 carbon atoms, such as, acetone,pentanone, 2-methyl pentanone hexanone, methyl ethyl ketone, methylbutyl ketone and/or heptanone; alcohols having 1 to 5 carbon atoms, suchas methanol, ethanol, n-propanol, 2-propanol, n-butanol, 2-butanol,n-pentanol and tert-butanol; halogenated solvents, such asdichloromethane, 1,2-dichloroethane, chloroform, carbon tetrachloride,tricholoroethane, 1,2-dichloropropane or 1,2,3-trichloropropane oraromatic solvents, such as benzene, phenyl, toluene and p-methyl tolueneas well as mixtures thereof. The polycosanols are selectively extractedwith the organic solvent to provide a polycosanol composition of higherpurity.

In one embodiment of the present invention, the polycosanol productobtained from the extraction is further purified by means ofrecrystallization.

Included in the present invention are the polycosanol compositions ofmatter prepared and isolated by the method of this invention. The longchain aliphatic alcohol portions of the polycosanol compositionsprepared and isolated by the methods of this invention are comprisedprimarily of the four primary aliphatic alcohols 1-hexacosanol(20%-50%), 1-octacosanol (15%-45%), 1-triacontanol (2%-10%) and1-tetracosanol (approximately 1%-9%). The total amount of long chainaliphatic alcohols in these compositions can be anywhere in the range of35% to 100%, depending on the level of purification of the crude extractobtained from step (b) of the described method. In one embodiment, thecomposition of matter is isolated following hydrolysis of the wax andneutralization with no further purification. This composition iscomprised of approximately 35-55% of the long chain primary aliphaticalcohols of interest. The major components of this composition are:1-hexacosanol (˜20-30%), 1-octacosanol (˜15-25%), with two minorcompounds: 1-triacontanol (˜2-4%) and 1-tetracosanol (˜1-3%). In anotherembodiment, the composition of matter is isolated following hydrolysisof the wax, without neutralization and further purification viaextraction with an organic solvent. In this embodiment the compositionof matter is comprised of approximately 75-100% of the long chainprimary aliphatic alcohols. The major components of this composition are1-hexacosanol (˜30-50%), 1-octacosanol (˜25-45%), with two minorpolycosanols: 1-triacontanol (˜4-10%) and 1-tetracosanol (˜3-9%). Asnoted above, the alcoholic mixtures obtained from Ericerus pela wax inaccordance with the present invention can be distinguished from allother currently known sources of polycosanols. Table 1, below highlightsthe differences between the compositions isolated from sugar cane andbee wax, which are currently the major commercially available sources ofpolycosanols, with the composition isolated according to the method ofthis invention. With reference to Table 1, it can be seen that thecomposition of matter isolated from E. pela has a much greaterpercentage of hexacosanol (C26) (˜45%) than either sugar cane (˜6-9%) orbee wax (˜7-12%). The polycosanol isolated from E. pela also has asomewhat lower percentage of triacontanol (C30).

Further included in this invention is the use of the compositions ofmatter produced for the prevention and treatment of obesity, syndrome X,diabetes, hypercholesterolemia, atherosclerotic complications, ischemiaand thrombosis.

Various delivery systems are known in the art and can be used toadminister the therapeutic compositions of the invention, e.g., aqueoussolution, encapsulation in liposomes, microparticles, and microcapsules.

Therapeutic compositions of the invention may be administeredparenterally by injection, although other effective administrationforms, such as intraarticular injection, inhalant mists, orally activeformulations, transdermal iontophoresis or suppositories are alsoenvisioned. One preferred carrier is physiological saline solution, butit is contemplated that other pharmaceutically acceptable carriers mayalso be used. In one preferred embodiment, it is envisioned that thecarrier and polycosanol composition constitute aphysiologically-compatible, slow release formulation. The primarysolvent in such a carrier may be either aqueous or non-aqueous innature. In addition, the carrier may contain otherpharmacologically-acceptable excipients for modifying or maintaining thepH, osmolarity, viscosity, clarity, color, sterility, stability, rate ofdissolution, or odor of the formulation. Similarly, the carrier maycontain still other pharmacologically-acceptable excipients formodifying or maintaining the stability, rate of dissolution, release orabsorption of the ligand. Such excipients are those substances usuallyand customarily employed to formulate dosages for parentaladministration in either unit dose or multi-dose form.

Once the therapeutic composition has been formulated, it may be storedin sterile vials as a solution, suspension, gel, emulsion, solid, ordehydrated or lyophilized powder; or directly capsulated and/or tabletedwith other inert carriers for oral administration. Such formulations maybe stored either in a ready to use form or requiring reconstitutionimmediately prior to administration. The manner of administeringformulations containing the compositions for systemic delivery may bevia enteral, subcutaneous, intramuscular, intravenous, intranasal orvaginal or rectal suppository.

The amount of the composition that will be effective in the treatment ofa particular disorder or condition will depend on the nature of thedisorder of condition, which can be determined by standard clinicaltechniques. In addition, in vitro or in vivo assays may optionally beemployed to help identify optimal dosage ranges. The precise dose to beemployed in the formulation will also depend on the route ofadministration, and the seriousness or advancement of the disease orcondition, and should be decided according to the practitioner and eachpatient's circumstances. Effective doses may be extrapolated fromdose-response curved derived from in vitro or animal model test systems.For example, an effective amount of the composition of the invention isreadily determined by administering graded doses of the composition ofthe invention and observing the desired effect.

The method of treatment according to this invention comprisesadministering internally or topically to a host in need thereof atherapeutically effective amount of the polycosanol composition isolatedaccording to the method of this invention. The purity of the polycosanolcomposition administered includes, but is not limited to 0.01% to 100%,depending on the methodology used to obtain the compound(s). In apreferred embodiment doses of the polycosanol and pharmaceuticalcompositions containing that same are an efficacious, nontoxic quantitygenerally selected from the range of 0.01 to 200 mg/kg of body weight.Persons skilled in the art using routine clinical testing are able todetermine optimum doses for the particular ailment being treated.

This invention includes an improved method for isolating and purifyingpolycosanol compositions from a novel source, the wax of the insect E.pela. 1 describes gas chromatographic (GC) method used to quantify thevarious polycosanols present in the compositions isolated. Examples 2-6describe various methods used to hydrolyze/saponify the wax and theprofile of the composition obtained using each method. Examples 7-12describe various methods for further purifying the initial hydrolyzedextract isolated and the profile of the composition obtained using eachmethod. Example 13 describes an experiment performed to determine theeffect of the composition of this invention on lipid levels. Withreference to Table 13, it can be seen that HDL levels were raisedsignificantly in the mice treated with both 2.7 mg/kg/day (15 mgequivalent human dose, p<0.05) and 5.5 mg/kg/day (30 mg equivalent humandose, p<0.01) over a six month period. The data also shows that totalcholesterol and LDL levels were reduced. TABLE 1 Composition ofpolycosanol from different natural sources Sugar Cane Wax Bee Wax InsectWax Existing Form Primarily esters Free alcohols and esters EstersHydrolysis Required Not necessarily required Required Alcohol Percentage(w/w %) Percentage (w/w %) Percentage (w/w %) 1-tetracosanol 0.5-1   1-43-5 1-hexacosanol 5.5-8.5  7-12 44-46 1-heptacosanol   2-3.5 1-4 01-octacosanol 60-70 30-60 35-40 1-nonacosanol 0.4-1.2 2-5 01-triacontanol 10-15 16-26 4-8 1-dotriacontanol 4-6 13-22 Trace1-tetratriacontanol 0.4-2   2-6 0

The following examples are provided for illustrative purposes only andare not intended to limit the scope of the invention.

EXAMPLES Example 1 Quantification of Polycosanol using GasChromatography

The method for quantifying polycosanol was developed using a Shimazu GasChromatograph (GC-17A). The separation was carried out on one of thefollowing GC columns: XTI-5 (bonded 5% phenyl, 30 meters, 0.25 mm ID and0.5 μm thick) or DB-5HT (fused silica gel, 30 meters, 0.25 mm ID and 0.1μm film). The carrying gas was helium at a flow rate 3.0 mL/min. Thetemperature settings were as follows: injector 375° C., detector 375°C., column oven starting at 190° C. (1 minute) and increasing to 315° C.at the rate of 35° C./minute. The column oven temperature was then keptat 315° C. for 10 minutes. Sample and standards were dissolved in THF ata concentration between 50 to 200 ng/μL and directly injected onto thetop of column without splitting at a volume of 2 μL per injection. Theeluted alcohols were detected with a FID detector. Compoundidentification was based on retention time derived from individualalcohol standards. A calibration curve was measured for each standard atseven different concentrations (50, 100, 150, 250, 400, 600 and 800 ng).For wax sample quantification, hexacosanol was utilized as an externalstandard and response factors were calculated based on theconcentrations and GC peak area as 0.62/0.94/1.0/1.25/1.98(C24/C26/C27/C28/C30).

Example 2 Hydrolysis of Ericerus pela Insect Wax in Ethanol

Ericerus pela insect wax (25 g) was melted at a temperature between 80to 100° C. Sodium hydroxide solution (5 g dissolved in 5 mL of water)was added to the wax with 95 mL of 30% ethanol. The mixture was stirredand refluxed for 4 hours and then allowed to cool to room temperature.The white solid obtained was filtered and washed twice with water (500mL), then neutralized with glacial acetic acid (5 mL) and washed withwater (3×) until neutral. As much as 22.5 g of crude polycosanol wasobtained at a yield of 90%, containing four major fatty alcohols(1-tetracosanol, 1-hexacosanol, 1-octacosanol and 1-triaconsanol) havinga purity of 62.2%. The alcohol profile of the product is set forth inTable 2 below. TABLE 2 Long chain alcohol profile of polycosanol product(hydrolysis in EtOH) Component Percentage (w/w %) 1-tetracosanol 2.71-hexacosanol 30.1 1-octacosanol 24.5 1-triacontanol 4.9

Example 3 Hydrolysis of Ericerus pela Insect Wax in Methanol

Ericerus pela insect wax (100 g) was melted at a temperature between 80to 100° C. Sodium hydroxide solution (25 g dissolved in 30 mL of water)was added into the wax with 290 mL of 52.5% methanol. The saponificationreaction was allowed to proceed for a period of five hours with heatingand stirring. The mixture was cooled to room temperature and filtered toobtain a white solid. The solid was washed with water (2×), neutralizedwith sulfuric acid (50 mL, 20%, w/w) and washed with water (3×). Thepolycosanol product (91 g) was obtained at a yield 91% having a purityof 59.0% based on four major fatty alcohols (1-tetracosanol,1-hexacosanol, 1-octacosanol and 1-triaconsanol). The alcohol profile ofthe product is set forth in Table 3 below. TABLE 3 Long chain alcoholprofile of polycosanol product (hydrolysis in MeOH) Component Percentage(w/w %) 1-tetracosanol 2.5 1-hexacosanol 28.3 1-octacosanol 24.11-triacontanol 4.1

Example 4 Hydrolysis of Ericerus pela Insect Wax in 1-Propanol

Ericerus pela insect wax (500 g) was melted at a temperature between 80to 100° C. Sodium hydroxide solution (100 g dissolved in 100 mL ofwater) was added to the wax with 1900 mL of 50% 1-propanol. Thesaponification reaction was allowed to proceed for a period of six hourswith stirring and heating. The mixture was cooled to room temperatureand filtered to obtain a white solid. The solid was washed with water(2×), neutralized with hydrochloric acid (300 mL, 25%, w/w) and washedwith water until neutral. A total of 460 g of polycosanol was obtainedat a yield of 92% having a purity of 55.0% based on four major fattyalcohols (1-tetracosanol, 1-hexacosanol, 1-octacosanol and1-triaconsanol). The alcohol profile of the product is set forth inTable 4 below. TABLE 4 Long chain alcohol profile of polycosanol product(hydrolysis in 1-PrOH) Component Percentage (w/w %) 1-tetracosanol 1.91-hexacosanol 26.7 1-octacosanol 22.9 1-triacontanol 3.5

Example 5 Hydrolysis of Ericerus pela Insect Wax in Water

Ericerus pela insect wax (100 g) was melted at a temperature between 80to 100° C. Sodium hydroxide solution (100 mL, 20%) was added and thereaction mixture was heated with stirring for 4 hours. After 4 hours thereaction mixture was cooled to room temperature and extracted (6×) in aSoxlet extractor using chloroform as the solvent. The combinedchloroform solution was evaporated to provide a purified polycosanol (35g, 35% yield). The product contained four major fatty alcohols(1-tetracosanol, 1-hexacosanol, 1-octacosanol and 1-triaconsanol) at atotal concentration of 93.2%. The alcohol profile of the product is setforth below in Table 5. TABLE 5 Long chain alcohol profile ofpolycosanol product (hydrolysis in H₂O) Component Percentage (w/w %)1-tetracosanol 4.1 1-hexacosanol 45.6 1-octacosanol 36.2 1-triacontanol7.3

Example 6 Hydrolysis of Ericerus pela Insect Wax in n-Butanol

Ericerus pela insect wax (25 g) was melted at a temperature between 80to 100° C. Sodium hydroxide solution (7.5 g of sodium hydroxidedissolved in 5 mL of water) was added with 95 mL of 52.5% n-butanol. Thesaponification reaction was allowed to proceed for a period of 4.5 hourswith stirring and heating. The mixture was cooled to room temperatureand filtered to obtain a white solid. The solid was washed with water(2×), neutralized with concentrated phosphoric acid (5 mL) and washedwith water (3×) until neutral. A total of 22 g of polycosanol (88%yield) was obtained having a purity of 60.7% based on four major fattyalcohols (1-tetracosanol, 1-hexacosanol, 1-octacosanol and1-triaconsanol) as shown in Table 6. TABLE 6 Long chain alcohol profileof polycosanol product (hydrolysis in n-BuOH) Component Percentage (w/w%) 1-tetracosanol 2.2 1-hexacosanol 29.4 1-octacosanol 25.11-triacontanol 4.0

Example 7 Isolation of Polycosanol from Ericerus pela Insect Wax byExtraction with Ethyl Acetate

Ericerus pela insect wax (25 g) was melted at 80-100° C. Potassiumhydroxide (5 g) dissolved in 300 mL of 25% ethanol in water was added tothe melted wax. The reaction mixture was maintained at 80-100° C. for4.5 hours with stirring and heating. The basic reaction solution wasthen cooled to room temperature and extracted with ethyl acetate (10×).The ethyl acetate extracts were combined and evaporated to providepurified polycosanol (6 g, 24% yield), containing four major fattyalcohols (1-tetracosanol, 1-hexacosanol, 1-octacosanol and1-triaconsanol) having a purity of 95.0%. The alcohol profile of theproduct is set forth in Table 7 below. TABLE 7 Long chain alcoholprofile of polycosanol product (EtOAc extraction) Component Percentage(w/w %) 1-tetracosanol 4.2 1-hexacosanol 43.7 1-octacosanol 39.91-triacontanol 7.2

Example 8 Isolation of Polycosanol from Ericerus pela Insect Wax byExtraction with Hexane

Ericerus pela insect wax (500 g) was melted at 100-105° C. Sodiumhydroxide (150 g) dissolved in 1500 mL of water was added to the meltedwax and the solution was heated with stirring for 5 hours. After fivehours a white solid was obtained by filtration. The solid was extractedfor 12 hours in a Soxlet extractor using hexane as the solvent. Thehexane solution was cooled to room temperature, resulting in thecrystallization of the polycosanol product. The crystallized product wasthen filtered and recrystallized in methanol. Upon recrystallizationpolycosanol (165 g, 33% yield) was obtained, having a purity 92.3%aliphatic alcohols. The melting point of the mixture was 85-91° C. Thefatty acid profile of this product is set forth in Table 8 below. TABLE8 Long chain alcohol profile of polycosanol product (hexane extraction)Component Percentage (w/w %) 1-tetracosanol 4.0 1-hexacosanol 45.81-octacosanol 38.3 1-triacontanol 4.0

Example 9 Isolation of Polycosanol from Ericerus pela Insect Wax byExtraction with Chloroform

Ericerus pela insect wax (200 g) was melted at 80-100° C. Sodiumhydroxide (60 g) dissolved in 500 mL of water was added to the meltedwax and the reaction was allowed to proceed for a period of 4 hours withstirring and heating. The solid obtained after the reaction wascompleted, was filtered and extracted for 10 hours in a solid-liquidextraction system using chloroform as the solvent. The solution wascooled to room temperature, resulting in the crystallization of thepolycosanol. The crystallized product was then filtered andrecrystallized in methanol/chloroform (3/1) mixture. Polycosanol (75 g,37.5% yield) was obtained having a purity of 93.2%. The melting point ofthe mixture ranged from 86 to 90° C. Table 9 sets forth the compositionof the polycosanol product. TABLE 9 Long chain alcohol profile ofpolycosanol product (CHCl₃ extraction) Component Percentage (w/w %)1-tetracosanol 4.1 1-hexacosanol 42.4 1-octacosanol 39.7 1-triacontanol7.0

Example 10 Isolation of Polycosanol from Ericerus pela Insect Wax byExtraction with Tetrahydrofuran (THF)

Ericerus pela insect wax (1000 g) was melted at 90-110° C. and potassiumhydroxide (300 g) dissolved in 1500 mL of water was added. Thesaponification reaction was allowed to proceed for 120 minutes withstirring and heating. A white solid was obtained, which was filteredfrom the reaction mixture and then extracted with THF in a solid-liquidextracting system. The THF extract was evaporated and the residual solidwas crystallized in petroleum ether to yield 355 g of polycosanol (35.5%yield) having a purity of 89.4%. The melting point of the mixture was 81to 85° C. Table 10 sets forth the alcohol composition of the product.TABLE 10 Long chain alcohol profile of polycosanol product (THFextraction) Component Percentage (w/w %) 1-tetracosanol 4.21-hexacosanol 43.5 1-octacosanol 37.2 1-triacontanol 4.4

Example 11 Isolation of Polycosanol from Ericerus pela Insect Wax byExtraction with Methylene Chloride

Ericerus pela insect wax (500 g) was melted at 90-100° C. and sodiumhydroxide (120 g) dissolved in 1000 mL of water was added. The reactionmixture was stirred and heated for a period of 6 hours. The white solidproduced was filtered and extracted with methylene chloride for a periodof 12 hours in a conventional solid-liquid extraction system. Theextraction solution was cooled to room temperature and the solidobtained was recrystallized in ethanol to yield 143 g of polycosanol(28.6% yield). The crystallized product contained total 91.8% fattyalcohols, having a melting point between 83-86° C. Table 11 shows theprofile of the polycosanol product. TABLE 11 Long chain alcohol profileof polycosanol product (CH₂Cl₂ extraction) Component Percentage (w/w %)1-tetracosanol 4.8 1-hexacosanol 41.4 1-octacosanol 40.2 1-triacontanol5.4

Example 12 Isolation of Polycosanol from Ericerus pela Insect Wax byExtraction with Petroleum Ether

Ericerus pela insect wax (500 g) was melted at 100-110° C. and sodiumhydroxide (150 g) dissolved in 700 mL of water was added. The mixturewas stirred and heated for 3 hours and then filtered. The solid obtainedwas extracted with petroleum ether for 16 hours in a solid-liquidextractor and the extraction solution was then cooled to roomtemperature and filtered. The solid was crystallized to yieldpolycosanol (150 g, 30% yield) having a purity of 95.0%. The meltingpoint of the mixture ranged from 83-87° C. Table 12 shows the long chainaliphatic alcohol profile of the crystallized product. TABLE 12 Longchain alcohol profile of polycosanol product (pet. ether extraction)Component Percentage (w/w %) 1-tetracosanol 4.7 1-hexacosanol 43.41-octacosanol 41.5 1-triacontanol 5.4

Example 13 Effect of Polycosanol Composition on Lipid Levels

Animal Model: C57BL/J-6 (Daihan BioLink, Korea) mice fed a high fat,cholesterol and cholic acid diet (TD-88051, supplemented with 15.8% fat,1.25% cholesterol, and 0.5% Na-cholate, Harlan Teklad, USA). The lipidprofile (mg/dl) in the high-fat diet induced hyperlipidemic C57BL/6Jmice (n=5/group) following treatment for 6 months is set forth in Table13. TABLE 13 Lipid Profile Following Treatment with PolycosanolComposition Groups* Cholesterol Triglycerides HDL-cholesterolLDL-cholesterol Control 366.20 ± 71.06 73.80 ± 19.52 56.00 ± 8.34  84.80± 20.04 Poli-90(2.7) 336.00 ± 52.23 71.60 ± 17.56  69.00 ± 11.42** 82.00± 12.14 Poli-90(5.5) 340.00 ± 34.92 71.80 ± 20.39  80.60 ± 6.58*** 67.20± 10.18*Poli-90(2.7), Polycosanol 90%, 2.7 mg/kg/day, orally for 6 months.*Poli-90(5.5), Polycosanol 90%, 5.5 mg/kg/day, orally for 6 months.# p value (Student's t-test)**p < 0.05 compared to control group***p < 0.01 compared to control group

1. A polycosanol composition of matter comprised of long chain aliphaticalcohols, wherein said long chain alcohols comprise 1-hexacosanol(20%-50%), 1-octacosanol (15%-45%), 1-triacontanol (2%-10%) and1-tetracosanol (1%-9%).
 2. The composition of claim 1, wherein saidcomposition is derived from the wax of the insect Ericerus pela.
 3. Thecomposition of claim 1 wherein said long chain aliphatic alcoholscomprise 35%-100% of said composition.
 4. The composition of claim 1wherein said long chain aliphatic alcohols comprise 35%-55% of saidcomposition.
 5. The composition of claim 1 wherein said long chainaliphatic alcohols comprise 75%-100% of said composition.